insects

Article Cleonis pigra (Scopoli, 1763) (Coleoptera: Curculionidae: Lixinae): Morphological Re-Description of the Immature Stages, Keys, Tribal Comparisons and Biology

Jiˇrí Skuhrovec 1,* , Semyon Volovnik 2, Rafał Gosik 3, Robert Stejskal 4 and Filip Trnka 5 1 Group Function of Invertebrate and Plant Biodiversity in Agro-Ecosystems, Crop Research Institute, Drnovská 507, CZ-161 06 Praha 6 Ruzynˇe,Czech Republic 2 Independent Researcher, 72311 Melitopol, Ukraine; [email protected] 3 Department of Zoology and Plant Protection, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland; cossonus@gmail.com 4 Administration of Podyji National Park, Na Vyhlídce 5, CZ-669 02 Znojmo, Czech Republic; [email protected] 5 Department of Ecology & Environmental Sciences, Faculty of Science, Palacký University Olomouc, Šlechtitel ˚u27, CZ-783 71 Olomouc, Czech Republic; fi[email protected] * Correspondence: [email protected]; Tel.: +420-702087694



Received: 19 August 2019; Accepted: 24 September 2019; Published: 30 September 2019


Abstract: Mature larvae and pupae of Cleonis pigra (Scopoli, 1763) (Curculionidae: Lixinae: Cleonini) are morphologically described in detail for the first time and compared with known larvae and pupae of other Cleonini species. The results of measurements and characteristics most typical for larvae and pupae of Cleonini are newly extracted and critically discussed, along with some records given previously. Keys for the determination of selected Cleonini species based on their larval and pupal characteristics are attached. Dyar’s law was used for the estimation of a number of larval instars of C. pigra. Descriptions of habitats, adult behavior, host plants, life cycle, and biotic interactions are reported here. Adults and larvae feed on plants from the Asteraceae family only (genera Carduus, Cirsium, Centaurea, and Onopordum). Oviposition occurs on the base of the plant stem or the root neck. In the process of larval development, a fusiform gall forms. C. pigra and Cyphocleonus achates can coexist in the same locality. In open habitats, the weevils become the prey of carnivorous animals.

Keywords: Coleoptera; Curculionidae; Lixinae; Cleonis; morphology; larva; pupa; biology; host plant; life history

1. Introduction The tribe Cleonini belongs to the subfamily Lixinae [1], together with two other tribes: Lixini (approximately 40 genera, see [2]) and Rhinocyllini (two genera; sometimes part of Lixini, see [3]). Currently, Cleonini weevils include approximately 97 valid genus-group taxa [2,4] and 546 valid species [5–7]. The distribution is known to be mainly in the Northern Hemisphere; from south of the equator, they are known only in continental Africa and Madagascar [2]. Representatives of this tribe prefer xeric habitats and sandy soil. Their larvae are mono, oligo or polyphagous on herbs and shrubs, and they develop mainly in the lower parts of the host plant, especially the roots or, rarely, lower stems [2]. Endophagous larvae develop inside the plant tissue of the root neck or the collar of the host plants or create a characteristically swollen gall on the root of the host plants [8,9]. The morphology of immature Cleonini is still poorly known, but the first detailed, illustrated larval descriptions were recently made [9–11].

Insects 2019, 10, 325; doi:10.3390/insects10100325 www.mdpi.com/journal/insects Insects 2019, 10, 325 2 of 25

The members of the tribe Cleonini are known as a potentially significant pest of cultivated plants (beets, spinach, mayweeds, etc.), and they have the potential to be used as biocontrol agents against invasive Palaearctic plants [2]. For example, Cyphocleonus achates (Fåhraeus, 1842) has been used to control invasive spotted knapweed (Centaurea stoebe L., Asteraceae) in North America [12,13]. Another situation is known from Australia, where Pachycerus segnis (Germar, 1824) was tested for use against the invasive plant Heliotropium europaeum L. (Boraginaceae; as P. cordiger,[14]). Among the members of the tribe Cleonini, there are some other potential biological control agents, such as the weevil Adosomus roridus (Pallas, 1781), used against Tanacetum vulgare L., but this would be less effective and potentially dangerous due to its oligophagy [9]. Introducing and using such species as biological control agents might be risky for native fauna/flora, similar to the introduction of the weevil Rhinocyllus conicus (Froelich, 1792) [15]. The weevil Cleonis pigra (Scopoli, 1763) is a trans-Palaearctic species distributed from the Iberian Peninsula to the Far East [16]. It is also known from Central India [17]. The most northern areas of its range in Europe are southern Finland [18] and southern Norway (Ringerike, 60◦1201900 N; the Natural History Museum, University of Oslo). The most northern records of its appearance in Asia are approximately 60–63◦ N in Sakha (Yakutiya), Russia [19]. The beetle is common all over Ukraine [20] and in central and southern Europe, but is rather rare in the northern parts of the European distribution. However, this species was also registered in the Red List of Norway as vulnerable [21] and in the Red List of Finland [22]. In 1919, C. pigra was recorded in North America for the first time [23]. Recently, it has been recorded in a relatively small area in the northeastern USA and southeastern Canada, in the Atlantic region and nearby [23]. Accordingly, there is believed to be an adventive species in North America [23–26]. The immature stages of C. pigra were described by Cawthra [25] and Scherf [27], but setal nomenclature and morphological terms are not well understood, and some details in chaetotaxy and drawings are still missing. Adult C. pigra were released in a testing pasture in Ontario, Canada, to decrease the population of Cirsium arvense (L.) Scop. [28], but further work has not been ongoing. The wide host range of C. pigra also includes the globe artichoke (Cynara cardunculus scolymus (L.) Hegi) [29,30], which is cultivated in many areas, including North America. Therefore, the beetle has not been considered a suitable biocontrol agent [31]. On the other hand, C. pigra is known as a pest of milk thistle (Silybum marianum (L.) Gaertn.), the seeds of which are important raw materials for pharmaceutics [32]. Incidentally, adults also feed on the sprouts of sunflowers (Helianthus annuus L. [33]) and non-Asteraceae plants; namely, Siberian pea shrub (Caragana arborescens Lam., Fabaceae; [34]), and beets (Beta vulgaris L., Amaranthaceae; see review [35]). Usually, all these damages occur only in spring and are insubstantial. Obviously, all data on the economic damage caused by C. pigra in beet plantations are based on misidentifications [35]. The main aims of this study are the following: (1) to re-describe larvae and pupae of Cleonis pigra in detail for the first time; (2) to compare this species with other known immature stages of this tribe; (3) to determine the number of larval instars via morphometric measurements; and (4) to provide details on their life history based on observations in central Europe.

2. Materials and Methods

2.1. Insect Collection and Laboratory Breeding The material used to describe the larvae and the pupae was collected, and field observations were conducted in Ukraine in the following localities:

(1) Sheep pasture on the alluvial floodplain, near the Kamyana Mohyla Reserve (46◦5700100 N, 35◦2801200 E). Altitude: up to 11 m.a.s.l. Bedrock: sandy chernozem. Dominant plant species: Cirsium ukranicum Besser ex DC., and Carduus hamulosus Ehrh. (syn. C. pseudocollinus (Schmalh.) Klokov.), with occasional trees of Elaeagnus commutata Bernh. ex Rydb. growing among the grass Insects 2019, 10, 325 3 of 25

(i.e., Echinops ritro subsp. ruthenicus L. (M. Bieb.) Nyman (syn. E. ruthenicus), and Centaurea adpressa Ledeb. ex Steud., Melilotus albus Medik.) in the clearings.

(2) Pishchanska Balka near Melitopol (46◦49050” N, 35◦20018” E). Altitude: up to 22 m.a.s.l. Bedrock: sandy chernozem with herbaceous covering. Dominant plant species: Calamagrostis epigeios (L.) Roth, Linaria genistifolia (L.) Mill., Artemisia absinthium L., Echium vulgare L., Sisymbrium loeselii L., Achillea millefolium L., Hieracium umbellatum L., Melilotus albus Medik., Centaurea diffusa Lam., Cirsium arvense (L.) Scop. (syn. C. setosum (Willd.) Besser), and Chenopodium urbicum L.

In the abovementioned localities, the life cycle, including feeding of adults and oviposition, was observed directly during the vegetation growing seasons of Carduus hamulosus, Cirsium ukranicum, and Centaurea diffusa from 2015 to 2017. The author S.V. collected all larvae and pupae of C. pigra within roots of Cirsium ukranicum. Some stems and roots (n = 37) were dissected to investigate preimaginal development, and a further 150 plants were dissected to determine the quantity of preimaginal specimens of C. pigra. All photographs in the field were taken with a Canon PowerShot SX500 IS digital camera (Canon Inc., Ota,¯ Tokyo, Japan). Laboratory observations were conducted in Melitopol, Ukraine (46◦500 N, 35◦220 E). The measurements of stems and roots were performed with a slide caliper and ocular micrometer. The size of root galls was determined at the greatest diameter. Adults were weighed on the Jadever electronic scale JKD-250 (Jadever Scale Co., Ltd, Taipei, Taiwan). Geographical distribution and phenology were studied from several entomological collections, specifically, the Schmalhausen Institute of Zoology of National Academy of Sciences of Ukraine (Kyiv), the TG Shevchenko Kyiv the National University Zoological Museum, the Zoological Institute of the Russian Academy of Sciences (St. Petersburg), the VN Karazin Kharkiv National University Museum of Natural History, and Igor Maltsev’s (Odessa) and Sergey Suchkov’s (Melitopol) private collections. In total, more than 230 pinned specimens were studied. A virtual database of entomological collections of the Natural History Museum, University of Oslo (NHMUO) were used as well. Unpublished data recorded in the previous project “Lixinae of Ukrainian steppe” (1981–1985) were also used here. Adults of C. pigra were also collected close to the roots of Cirsium eriophorum (L.) Scop. in the central part of the Czech Republic in May 2014. The specimens were transported to the entomology laboratory at the Crop Research Institute (Prague, Czech Republic), and a breeding colony was established. Five male and female pairs were maintained in an insect chamber at 20 ◦C, with a relative humidity of 70% and a 12 h photoperiod. Females laid eggs, and after hatching, the young larvae were preserved in Pampel fixation liquid (see below).

2.2. Morphological Description A part of each of the larval and pupal materials were preserved in Pampel fixation liquid (see [36]) and used for the morphological descriptions. The remaining specimens are deposited in the collection of the Group Function of Invertebrate and Plant Biodiversity in Agro-Ecosystems of the Crop Research Institute (Prague, Czech Republic). The insect host plant was identified by a plant taxonomist. Insect slides were prepared according to the May [37] guidelines. The observations and measurements were conducted using a light microscope with calibrated oculars (Olympus BX 40, SZ 11, both Olympus, Shinjuku, Tokio Prefecture, Japan; and Nikon Eclipse 80i, Nikon, Minato, Tokio Prefecture, Japan). The following characteristics were measured for each larva: head width, length of the body (larvae fixed in a C-shape were measured in the middle of the segments in lateral view), and width of the body in the widest place (i.e., meso- and metathorax). For the pupae, the length and the width at the widest place were measured. The lengths of all setae are visible in the figures. Drawings were made with a drawing tablet (Intuos Pro S, Wacom, Saitama Prefecture, Japan), and the digital images were subsequently processed with Adobe Photoshop (Adobe Inc., San Jose, CA, Insects 2019, 10, 325 4 of 25

USA), Corel Photo-Paint 11 (Corel, Ottawa, ON, Canada), and/or GIMP 2 (GIMP Development Team, Charlotte, NC, USA). The numbers of setae of bilateral structures are given for one side. We used the terms and abbreviations for the setae of the mature larvae and pupae according to Scherf [27], May [37,38], and Marvaldi [39,40]. All morphological abbreviations used in text: Abd. I–X—abdominal segments 1–10, Th. I–III—thoracic segments 1–3, at—antenna, st—stemmata, lr—labral rods, ur—urogomphi; setae: als—anterolateral, ams—anteromedial, as—alar (larva), cls—clypeal, d—dorsal (pupal abdomen), des—dorsal (larval head), dms—dorsal malar, ds—discal (pupal prothorax), ds—dorsal (larval abdomen), eps—epipleural, eus—eusternal, fes—femoral, fs—frontal, les—lateral epicranial, ligs—ligular, lms—labral, l, ls—lateral, lsts—laterosternal, mbs—malar basiventral, mds—mandibular, mes—median, mpxs—maxillary palp, os—orbital, pas—postantennal, pda—pedal, pds—postdorsal, pls—posterolateral, pes—postepicranial, pfs—palpiferal, pms—postmental, prms—premental, prns—pronotal, prs—prodorsal, ps—pleural, rs—rostral, sls—super lateral, sos—super orbital, ss—spiracular, stps—stipal, sts—sternal, trs—trochanter, ts—terminal, v—ventral (pupa), ves—ventral epicranial, vms—ventral malar, vs—vertical.

3. Results and Discussion

3.1. The Morphology of Immature Stages of Cleonis pigra

3.1.1. Materials Examined

Larvae: Ukraine, Zaporizhia Province, near the Kamyana Mohyla Reserve (46◦57001” N, 35◦2801200 E). Three mature larvae, collected on 12 August 2015 in the galls on the root necks of Cirsium ukranicum; Slovakia, Brestovany: one mature larva, 21 July 2012. Pupae: Ukraine, Zaporizhia Province, near the Kamyana Mohyla Reserve (46◦5700100 N, 35◦2801200 E). Four and three , collected on 12 August 2015 in the galls on the root necks of Cirsium ukranicum. ♂♂ ♀♀ 3.1.2. Description of Mature Larvae Measurements (in mm, n = 4). Body length: 14.8–17.2. Body width: (metathorax and abdominal segment I) 4.40–5.33. Head width: 2.45–2.80. General. Body stocky, slightly curved, rounded in the cross section (Figure1A). Cuticle without any spiculation. Coloration. Light brown or brown head with a distinct pale pattern around the frontal line (Figure1A). All thoracic and abdominal segments white; only dorsum of pronotum with elongated light brown stripe (Figures1A and2A). Vestiture. Setae thin; short to long; orange (Figure1A). Head capsule (Figure2A). Head suboval, slightly longer than wide, endocranial line weak, and as long as a one-third the length of frons. Frontal sutures on head distinct, but narrow; Y-shaped and extended to stemmata. Single stemma (st), in the form of a dark pigmented spot, located on each side anterolaterally. Des1 and des2 located in upper part of the central part of epicranium, des1 near to the middle part of epicranium, and des2 near to side of epicranium, des3 located anterially near to frontal suture, des4 located in the central part of epicranium, des5 located anterolaterally; all des very long, and almost all equal in length (Figure2A). Fs1 and fs2 placed medially, fs3 located anteriomedially, fs4 located anteriolaterally, and fs5 located laterally, close to the epistoma; fs1, fs3 and fs5 very long; fs2 and fs4 relatively long to long, as long as half-length of the remaining three setae (Figure2A). Les1–3 and ves1 as long as all des; and ves2 relatively long. Epicranial area with four pes in line with upper des2. Insects 2019, 10, 325 5 of 25

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Figure 1. Cleonis pigra mature larva, habitus. ( (AA)—shape)—shape of of the the body body (lateral (lateral view); view); ( (B)—lateral)—lateral view view of thoracic segments; ( C)—lateral view of abdominal segment II; (D)—lateral view of abdominal segments VII–X.VII–X. ( prns(prns—pronotal—pronotal seta(e), seta(e),prs —prodorsalprs—prodorsal s., pds s.,—postdorsal pds—postdorsal s., as—alar s., as s.,—alarss—spiracular s., ss— spiraculars., eps—epipleural s., eps—epipleural s., ps—pleural s., ps s.,—pleuralpda—pedal s., s.,pdalsts—pedal—laterosternal s., lsts—laterosternal s., eus—eusternal s., eus s.,—eusternalds—dorsal s., s., dssts—dorsal—sternal s., s., ststs—sternal—terminal s., s.; ts Th1–3—terminal and Ab1–10—numbers.; Th1–3 and Ab1–10—numbe of thoracic andr of abdominal thoracic and segments). abdominal segments). Antennae located at the end of the frontal suture on each side; membranous and convex basal articleAntennae bearing conicallocated triangularat the end sensorium,of the frontal very suture long; on basal each membranous side; membranous article with and fourconvex sensillae basal articledifferent bearing in both conical shape triangular and length sensorium, (Figure2D). very long; basal membranous article with four sensillae differentClypeus in both (Figure shape2E) and is approximatelylength (Figure 2.22D). times as wide as long with two relatively long to long cls; clsClypeus2 distinctly (Figure shorter 2E) thanis approximatelycls1, localized 2.2 posteriolaterally. times as wide Oneas long sensillum; with two anterior relatively margin long rounded to long clsto; the cls2 inside; distinctly median shorter part than covered cls1, localized by thorn-shaped posteriolaterally. cuticular One processes. sensillum; anterior margin rounded to the inside; median part covered by thorn-shaped cuticular processes.

Insects 2019, 10, 325 6 of 25 Insects 2019, 10, x 6 of 25

FigureFigure 2. 2.Cleonis Cleonis pigra pigramature mature larva, larva, head, head, antenna,antenna, and mouth parts. parts. (A (A)—dorsal)—dorsal view view (des (des—dorsal—dorsal epicranialepicranial seta seta (e), (e), fsfs—frontal—frontal epicranial epicranial s., s., lesles—lateral—lateral epicranial epicranial s., ves s.,—ventralves—ventral epicranial epicranial s., st— s., st—stemmata,stemmata, at—antenna); at—antenna); (B)—right (B)—right maxilla, maxilla, dorsal dorsalview (dms— viewdorsal (dms—dorsal malar s., vms— malarventral s., vms malar—ventral s., malarmpxs s.,—maxillarympxs—maxillary palps s., palps mbs—basoventral s., mbs—basoventral s., pfs—palpiferal s., pfs—palpiferal s., stps—stipital s., stps—stipital s.), prementum s.), prementum and andpostmentum, postmentum, ventral ventral view view (prms (prms—premental—premental s., s.,pmspms—postmental—postmental s., s.,ligsligs—ligular—ligular s.); s.); (C ()—rightC)—right mandiblemandible (mds (mds—mandible—mandible dorsal dorsal s.); s.); (D )—antenna;(D)—antenna; (E )—labrum(E)—labrum and and clypeus clypeus (cls —clypeal(cls—clypeal s., lmss., —labrallms— s.);labral (F)—epipharynx s.); (F)—epipharynx (ams—anteromedial (ams—anteromedial s., als—anterolateral s., als—anterolateral s., mes—median s., mes—median s., lr—labral s., lr—labral rods). rods). Mouth parts. Labrum (Figure2E) approximately 2.5 times as wide as long, with three piliform Mouth parts. Labrum (Figure 2E) approximately 2.5 times as wide as long, with three piliform lms, of different length; lms2 and lms3 distinctly shorter than very long lms1; lms1 is placed close to lms, of different length; lms2 and lms3 distinctly shorter than very long lms1; lms1 is placed close to the the margin with clypeus, lms2 is located anteriomedially, and lms3 is located anteriolaterally; anterior margin with clypeus, lms2 is located anteriomedially, and lms3 is located anteriolaterally; anterior margin double sinuate. Epipharynx (Figure2F) with four blunt, finger-like als, unequal in length; margin double sinuate. Epipharynx (Figure 2F) with four blunt, finger-like als, unequal in length; two two laterally als distinctly longer than two medially als; three ams, ams1, and ams2 blunt, finger-like, laterally als distinctly longer than two medially als; three ams, ams1, and ams2 blunt, finger-like, distinctly larger than piliform ams3; two very short to short, blunt mes, unequal in length, both located

Insects 2019, 10, 325 7 of 25

close to lr; and one sensillum close to mes2; labral rods (lr) elongated, triangularly, converging anterially. Mandibles (Figure2C) distinctly broad, bifid, tooth of unequal height; slightly truncate; both mds relatively long, hairform, located in distinct holes. Maxilla (Figure2B) stipes with one stps, two pfs and one mbs; stps and pfs1-2 very long, equal in length; mbs very short. Mala with 11 dms in two different lengths (six bacilliform relatively long and five piliform long to very long); five vms, four as long as bacilliform dms, and one very short. Maxillary palpi with two palpomeres: basal palpomere with one very short mxps and two sensilla; length ratio of basal and distal palpomeres: 1:0.6; distal palpomere with one sensillum and a group of conical, cuticular apical processes. Praelabium (Figure2B) heart-shaped and partially elongated, with one very long prms; ligula with sinuate margin and three hairform short ligs, unequal in length; premental sclerite, ψ-shaped, and well sclerotized. Labial palpi with two palpomeres; length ratio of basal and distal palpomeres: 1:0.5; distal palpomere with one sensillum and short, cuticular apical processes; basal palpomere with one dorsal sensillum. Postlabium (Figure2B) with three pms: pms1 located anterially, remaining two pairs laterally; pms1 and pms2 very long; pms3 as long as one third-length of the previous two setae. Surface of postlabium partly covered by distinct cuticular processes. Thorax. Prothorax distinctly smaller than meso and metathorax. Spiracle bicameral. Prothorax (Figure1B) with 11 prns unequal in length, eight of them on weakly pigmented dorsal sclerite; this sclerite is subdivided into two triangular plates medially, three of setae closely to the spiracle; two relatively long ps and two relatively long eus. Mesothorax (Figure1B) with one prs, five pds unequal in length; pds1 short to relatively long, pds2–3 and pds5 relatively long to long, pds4 short; one short to relatively long as; two ss equal in length, both short; one relatively long eps; one relatively long ps and two relatively long eus. Chaetotaxy of metathorax (Figure1B) almost identical to mesothoracal, metathorax with two or three ss almost equal in length, all short. Each pedal area of thoracic segments well separated, with six relatively long to long pda, and one short pda. Abdomen. Abdominal segments I–IV are of almost equal length, with subsequent abdominal segments decreasing gradually to the terminal parts of the body. Abdominal segment X reduced to four anal lobes of unequal size, the dorsal being distinctly the largest, the lateral pair equal in size, and the ventral lobe very small. Anus located terminally. Spiracles bicameral, the eight abdominal spiracles located laterally, close to the anterior margin of abdominal segments I–VIII. Abdominal segments I–VII (Figure1C) with one short prs; seven pds unequal in length, pds1–2, pds4, and pds6 short to very short, pds3, pds5 and pds7 long, but abdominal segment VII without pds1; one short to relatively long ss; two eps of unequal length, eps1 short, eps2 long; two ps of unequal length, ps1 short, ps2 long; one short lsts and two short eus. Abdominal segment VIII (Figure1D) with one short prs; five pds unequal in length, pds4,6 short to very short, pds3,5,7 long, pds1–2 absent; one short to relatively long ss; two eps of unequal length, eps1 short, eps2 long; two ps of unequal length, ps1 short, ps2 long; one short lsts and two short eus. Abdominal segment IX (Figure1D) with five ds (ds1,3,5 long and ds2,4 short); three ps of unequal length, ps1 very short to minute, but ps2–3 almost as long as ds1; and two very short to minute sts. Lateral anal lobe on abdominal segment X (Figure1D) with two very short to minute setae ( ts).

3.1.3. Description of Pupae Measurements (in mm; four , three ). Body lengths: 13.7–15.6 (mean 14.9) and 14.8–16.7 (mean 16.5). Body widths: 5.1–5.6♂♂ (mean♀♀ 5.5) and 6.0–7.5♂ (mean 6.7). Thorax widths:♀ 4.7–4.9 (mean 4.5) and 3.8–4.5 (mean♂ 4.5). Head widths: 2.2–2.3♀ (mean 2.2) and 2.3–2.4 (mean 2.3).♂ Coloration.♀ All thoracic and abdominal segments♂ light yellowish.♀ Cuticle smooth, except thorn-like processes on abdominal segments II–VIII. Morphology (Figure3A–C). Body slender and elongated. Rostrum long, about three times as long as wide, and extended to mesocoxae. Antennae rather short. Pronotum 1.4 times as wide as long. Mesonotum slightly shorter than metanotum. Abdominal segment I rather short; abdominal segments II–IV of equal length, longer than abdominal segment I; abdominal segment VI relatively long; abdominal segment VII almost semicircular; abdominal segments VIII and IX distinctly smaller Insects 2019, 10, 325 8 of 25 Insects 2019, 10, x 8 of 25 thanthan other other segments. segments. UrogomphiUrogomphi ratherrather short,short, coni conical,cal, with with sclerotized sclerotized apexes. apexes. Spiracles Spiracles placed placed laterally;laterally; on on abdominal abdominal segments segments I–VI–V functional,functional, and and on on abdominal abdominal segment segment VI VI atrophied; atrophied; on onnext next segmentssegments spiracles spiracles invisible. invisible. SexualSexual dimorphism visible visible in in the the structure structure of ofabdominal abdominal segment segment IX: IX: gonothecagonotheca of of ♂undivided undivided (Figure (Figure4 D),4D), ♀divided divided (Figure (Figure4 E).4E). ♂ ♀

FigureFigure 3. 3. CleonisCleonis pigra pigra pupa,pupa, habitus. habitus. (A)—dorsal (A)—dorsal view; (B view;)—ventral (B)—ventral view; (C)—lateral view; ( Cview)—lateral (Ab1–9— view (Ab1–9—numbernumber of abdominal of abdominal segments, segments, Th1–3—number Th1–3—number of thoracic of segments, thoracic segments,ur—urogomphi). ur—urogomphi).

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FigureFigure 4. Cleonis4. Cleonis pigra pigrapupa, pupa, chaetotaxy. chaetotaxy. ( AA)—head)—head and and rostrum; rostrum; (B)—lateral (B)—lateral view view of head of headand and pronotum;pronotum; (C)—dorsal (C)—dorsal view; view; (D )—ventral(D)—ventral view view of of last last abdominal abdominal segments segments of of male; male;( E(E)—ventral)—ventral view view of last abdominal segments of female (Ab1–9—number of abdominal segments, Th1–3—number of last abdominal segments of female (Ab1–9—number of abdominal segments, Th1–3—number of thoracic segments, ur—urogomphies. Setae: l, ls—lateral, sls—super lateral, d—dorsal, ds—discal, of thoracic segments, ur—urogomphies. Setae: l, ls—lateral, sls—super lateral, d—dorsal, pls—posterolateral, trs—trochanters, v—ventral, vs—vertical, sos—super orbital, os—orbital, pas— ds—discal, pls—posterolateral, trs—trochanters, v—ventral, vs—vertical, sos—super orbital, os—orbital, postantennal, rs—rostral, fes—femoral). pas—postantennal, rs—rostral, fes—femoral).

Chaetotaxy (Figure4A–E). Setae distinct, with different lengths; hair-like; light yellow. Head capsule includes one vs; three sos equal in length; and one os and four pas equal in length. Rostrum with four rs, different in length: rs1 and rs2 very short and rs3 and rs4 long; rs1–3 located apically and rs4 latero-apically. Insects 2019, 10, 325 10 of 25

Pronotum with one as, three sls, two ds, four pls, and three ls; discal setae very short, remaining setae elongated, equal in length (Figure4B). Mesonotum with five setae ( d) of which: the first is located antero-medially; the next four form a diagonal line medially. Metanotum with five setae (d) forms a diagonal line medially. All setae on meso and metanotum are equal in length (Figure4C). Trochanter of prolegs with two trochanters setae (trs1–2) (Figure4A). Each apex of femora with three elongated fes (Figure4C). Abdominal segment I with nine short, hair-like setae of which: d1 is located antero-medially, d2–8 are along posterior margin of segment, d9 is located anterolaterally. Setae d2–7 on abdominal segments II–VII replaced by thorn-like cuticular processes. Setae d7 and d8 are alike; cuticular processes increase from abdominal segments II to VII, while d1 and d9 stay as short as those on abdominal segment I. Abdominal segment VIII has one very short antero-medially seta, three thorn-like cuticular processes (of which first distinctly longer than next), all distributed along posterior margin, and finally, three elongated postero-laterally setae placed on protuberances. Each of lateral parts of abdominal, segments I–VIII, have one very long seta. Each ventral part of abdominal segments I–VIII has four short setae, forming a regular, horizontal line on median parts. Abdominal segment IX has three short setae placed on urogomphi and next single setae on gonothecae.

3.1.4. Remarks on the Previous Descriptions of Immature Stages of Cleonis pigra The larval description of C. pigra by Scherf [27] used a different setal nomenclature and morphological terms, rendering understanding difficult. The chaetotaxy of the head described by Scherf [27] is questionable because he listed only four fs, three or four vms, and 12 dms. In contrast, we listed the presence of five fs, five vms, and 11 dms (Figure2A,B). Scherf [ 27] also listed that the labrum and epipharynx have nine setae, but we observed three lrms, three ams, four als, and two mes. Some of the setae on the epipharynx (especially ams and mes) cannot be compared exactly due to unclear drawings and unclear resolution of the distal mes, which some authors listed as ams (see [41]). The chaetotaxy of the body is more problematic due to missing drawings, and the count of setae is questionable (e.g., [27] listed 23 setae on the pronotum, but we observed only 22 setae). Cawthra [25] presented some drawings of the mature larva of C. pigra, but understanding them is problematic. The pupal description by Scherf [27] is confusing and different from our observations, he listed only one rs, two fes, eight setae on the pronotum, and six setae on the mesonotum. In contrast, we listed the presence of four rs, three fes, 13 setae on the pronotum, and five setae on the mesonotum (Figure4C).

3.1.5. A Comparison with Larvae of Other Cleonini The larvae of twelve Cleonine taxa have already been described in previous studies [9–11,27,42,43], and a detailed description of the pupae is known for only eleven Cleonine taxa [9–11,27,43]. The comparison of the larva and pupa of Cleonis pigra with those described by Hoffmann [42] and Scherf [27] was somewhat difficult due to the use of different terminology for morphology and chaetotaxy and/or the absence of good quality drawings (see [9]). Despite these challenges, we were able to compare the morphology of eleven known taxa (except description from [42]) and give tha comparison in the Key (see Key for the known immature stages of Cleonini). The mature larvae of the subfamily Lixinae were characterized by three diagnostic features [44]: the increased number of pds (1) on the meso- and metathorax and (2) abdominal segments I–VII, and (3) the increased number of setae on the epipharyngeal lining (als); i.e., more than the most frequent number of setae in weevils (for details, see [11]). These differential features of mature larvae were confirmed in all known descriptions from the tribe Cleonini [9–11,27,43], and all known species from the Lixini tribe fit this diagnosis (genus Larinus [45–48]; Lixus species [27,45,49–55]; and Rhinocyllus conicus [37]). The larval morphological features, such as (1) the presence of endocarina (Figure2A), (2) the count and position of setae on the labrum (Figure2E), and (3) the count of setae on the mandible (Figure2C), seem to be important features for generic study of the Cleonini. The key for all known immatures from Insects 2019, 10, 325 11 of 25 the tribe Cleoinini is presented below (see Table1). The detailed generic study of the Cleonini tribe and the comparison of both tribes is not still possible because of our limited knowledge of the immature stages. However, all these data confirm that the detailed descriptions of immature stages are very important for further studies on generic and suspected generic taxonomic relationships within Lixinae, as well as for the effective protection of endangered species, the exploitation of their potential in life science, and the promotion of using larvae of Cleonini species as potential biological control agents against weeds (e.g., Carduus, Cirsium, Tanacetum)[9]. Although the number remains low in comparison with the total number of Cleonini species, these results demonstrate the possibility of identifying the immature stages in these species, as was done in other groups of weevils (see Otiorhynchus [56], Tychiini [57–59], and Mecinini [60]).

3.1.6. Key to the Known Immature Stages of Cleonini

Larvae (Last Instar) The key is based on recently detailed descriptions of mature larvae of Cleonis pigra and ten descriptions of larvae published before [9–11,27,43] (Table1).

Pupae The key is based on recent descriptions of pupa of Cleonis pigra and descriptions of pupae published before [9–11,43] (Table2).

3.1.7. Measurements of Larval Instars Cawthra [25] and Scherf [27] concluded that larvae of C. pigra have four instars. We calculated the hypothetical cephalic width according to Dyar’s law (the observed model that increases in sclerotized body parts during development of the arthropod’s immature stages are predictable and normal by a relatively constant factor) using the ratios 1.35, 1.40, and 1.50 [61,62] because we were able to measure the head capsule only for the first instar and the last instar. The best approximation to the real size was obtained for four instars as in Scherf [27], but we had small discrepancies in the measured size in the first and fourth instars. Cawthra [25] listed the following ranges of head width of each instar: L1 0.77–0.92 mm; L2 1.08–1.31 mm, L3 1.54–1.93 mm, and L4 2.16–2.62 mm. Scherf [27] listed identical measurements as Cawthra [25], but he did not mention this paper. On the other hand, we measured a small head width for both instars: L1 0.99–1.17 mm and L4 2.45–2.80 mm.

3.2. Biology and Ecology of Cleonis pigra

3.2.1. Habitats The weevil Cleonis pigra occurs predominantly in coastal biotopes, forest edges and glades, man-made treelines, roadsides, quarries, wastelands and other ruderal plots, and pastures (Figure5A,B). Adults are usually recorded on the soil surface or in the upper soil level. Sometimes, they are locally rather numerous. They can be found under stones [63]. This weevil prefers biotopes with light sandy soils. In the southwest of Hungary, it inhabits the recultivated dumps of uranium mines [64]. In the Czech Republic, C. pigra prefers disturbed habitats, such as roadsides, fallow land, and waste places [65]. In Turkey, an adult was recorded as high as 2450 m.a.s.l. in the mountains [66]. Quite often, C. pigra is encountered in agricultural lands-fields [67], plantations, orchards, and vineyards [68]. In western Ukraine, C. pigra accounted for more than half of the total weevils recorded in sugar beet plantations [69]. Adults are attracted there by an abundance of Asteraceae weeds. As a result, numerous beetles of the new generation are found in combined oats [70,71] and in bagged and stored beans [70,72] during or after harvesting. Insects 2019, 10, 325 12 of 25

Table 1. Key to the Known Larvae of Cleonini.

1. Endrocarina present. 2 - Endrocarina absent. 9 2. Labrum with three lrms. 3 - Labrum with two lrms. Scaphomorphus erysimi 3. Setae on labrum (lrms) in a triangle position. 4 - Setae on labrum (lrms) in a line. 6 4. Dorsum on abdominal segments I–VII with six or more pds. Ligula with three setae. Mandibula with two setae. 5 - Dorsum on abdominal segments I–VII with four pds. Ligula with two setae. Mandibula with one seta. Asproparthenis carinicollis Dorsum on abdominal segments I–VII with six pds (not in one line) and two ss (one not in line with pds). 5. Adosomus roridus Dorsum on abdominal segment VIII with five pds and two ss. Dorsum on abdominal segments I–VII with seven pds and one ss (all in one line). - Cleonis pigra Dorsum on abdominal segment VIII with five pds and one ss. 6. Head with four des. 7 - Head with five des. 8 7. Two des more than twice as long as the remaining two setae Bothynoderes declivis - All four des of almost the same size Bothynoderes affinis 8. Mandibula with one seta. Maxilla with one mbs. Cyphocleonus achates - Mandibula with two setae. Maxilla without mbs. Rhabdorrhynchus karelinii 9. Setae on labrum (lrms) in a triangle position. 10 - Setae on labrum (lrms) in a line. Pachycerus scabrosus sensu Scherf (1964) 10. Mandibula with two setae. Epipharynx with four als. Ligula with two setae. Cyphocleonus dealbatus - Mandibula with one seta. Epipharynx with six als. Ligula with three setae. Coniocleonus nigrosuturatus Insects 2019, 10, 325 13 of 25

Table 2. Key to the Known Pupae of Cleonini.

Mesonotum with five or six setae. Dorsum of abdominal segments I–VI with nine or ten setae 1 2 (sometimes setae replaced by thorn-like asperities). Mesonotum with three setae. Dorsum of abdominal segments I–VI with six or eight setae - 6 (sometimes setae replaced by thorn-like asperities). 2. Head with two or three sos. 3 - Head with one sos. Mesonotum with five setae. Dorsum of abdominal segments I–VI with ten setae. Rhabdorrhynchus karelinii 3 Mesonotum with six setae. 4 Mesonotum with five setae. Rostrum with four rs. Dorsum of abdominal segments I–VI with nine setae. -. Cleonis pigra Each apex of femora with two fes. 4 Rostrum with one rs. Dorsum of abdominal segments I–VI with nine setae. 5 - Rostrum with two rs. Dorsum of abdominal segments I–VI with ten setae. Each apex of femora with two fes. Adosomus roridus 5. Pronotum with ten setae. Head with four pas. Each apex of femora with three fes. Coniocleonus nigrosuturatus - Pronotum with eleven setae. Head with five pas. Scaphomorphus erysimi 6 Head with two sos. Metanotum with three setae. 7 -. Head with one sos. Metanotum with four setae. Bothynoderes affinis 7. Dorsum of abdominal segments I–VI with six setae. Asproparthenis carinicollis - Dorsum of abdominal segments I–VI with eight setae. Cyphocleonus achates Insects 2019, 10, 325 14 of 25

3.2.2. Adult Behavior The weevil Cleonis pigra is active in the day. In April, motionless adults are often found on the places warmed by the sun. They feed on the leaves and are most active in warm dry weather. Usually, a feeding adult can be found on the lower surface of the leaf, near its margin. Without moving, the adult bites and rips pieces of mesophyll tissue of leaves and young sprouts. When the apex of its rostrum reaches the most distant point, the weevil begins to gnaw from the starting point again. This results in semicircular apertures in the leaves (Figure5C). If the leaf is large, the inner edges of apertures do not reach the midrib. The adult did not eat the small spines along the margins of the leaf (Cirsium, Carduus, and Onopordum). These spines fell down when the weevil ate the soft tissue around their base. Occasionally, a beetle creates this round hole in a leaf blade (Arctium). Cleonis pigra is capable of fasting for an average of 28 days [73]. When adults do not feed, they are found on the underside or on the base of leaves or near the base of the main stem (Cirsium, Carduus). In response to a slight disturbance, C. pigra beetles climb on the underside of the leaf they are currently on or fall to the ground and go into thanatosis. One of the common names of C. pigra is “sluggish weevil”. The color pattern of its covering makes it barely visible on the grey soil and plant debris, but does not hide it on the coastal sands (Figure5D,E). Therefore, these beetles often become victims of water birds (see below—Biotic interactions). To move from the upper side of the leaf to the lower side, the beetle goes to the leaf margin, moves its forelegs to the lower side and turns to the lower side in one sharp heave. C. pigra beetles have long wings and unfused elytra, but we have never seen them flying. Sometimes, an abruptly caught beetle releases a drop of semi-liquid greenish excrement.

3.2.3. Host Plant In Ukraine, we recorded both adults and larvae feeding on Cirsium ukranicum Besser ex DC., C. arvense (L.) Scop., Carduus nutans L., C. hamulosus Ehrh., C. uncinatus M. Bieb., Centaurea diffusa, C. odessana Prodan, and Onopordum acanthium L. Imago fed on Arctium L., Taraxacum F. H. Wigg., and Hieracium L. [35]. Isaev [74] reared imago from the root of Jurinea cyanoides (L.) Rchb. In the Czech Republic and Slovakia, frequent hosts of Cleonis pigra are Cirsium vulgare (Savi) Ten., Carduus acanthoides L., and Arctium species. Less often, the larvae were also found in Centaurea stoebe in southern Moravia (R. Stejskal, pers. observ.). According to Batra et al. [75], in the field, Cnicus L. and Silybum Vaill. were also recorded as host plants for C. pigra. In laboratory tests, beetles fed on the plants of 17 genera, exclusively Asteraceae [75]. Thus, it is oligophagous (first-degree oligophagy sensu [76]). In the field, imago prefers larger plants [77]. In northern Ukraine, over 90% of populations of Cirsium arvense and approximately 40% of the population of Onopordum acanthium were attacked by larvae of C. pigra [69]. In European populations, only 7% of Carduus nutans plants were attacked by C. pigra [78], but up to 18% [79] or even 80% [25] of Cirsium arvense plants were attacked by C. pigra. In Canada, beetles were recorded on thistle (Cirsium arvense) 12% of the time, on average [80]. High host plant density results in a high number and density of herbivores. The average density of C. pigra in the monoculture of Silybum marianum reaches almost 21 larvae on 25 plants [32]. Throughout spring to the end of summer, infection of plants by C. pigra significantly decreases because over 30% of immature instars die [25]. However, in small plots (up to 0.01 ha), the plant rate infestation, at times, may reach 100% [81]. If Cirsium arvense and Carduus tenuiflorus Curt. grow at the same locality, C. pigra prefers only the first [25]. Generally, it seems that Cirsium is the most preferable host plant [73]. According to Anderson [23], this weevil has no host races. Interestingly, there is no information about C. pigra on C. diffusa in North America where both species are adventive. Insects 2019, 10, 325 15 of 25

Insects 2019, 10, x 15 of 25

Figure 5. Cleonis pigra habitats. (A)—habitat with flowering Cirsium ukranicum (near the Kamyana Figure 5. Cleonis pigra habitats. (A)—habitat with flowering Cirsium ukranicum (near the Kamyana Mohyla Reserve); (B)—habitat with flowering Centaurea diffusa (Pishchanska Balka); (C)—the schema Mohyla Reserve); (B)—habitat with flowering Centaurea diffusa (Pishchanska Balka); (C)—the schema how adult of Cleonis pigra eats a leaf of Cirsium setosum. Asterisks mark the beetle location; (D)— how adult of Cleonis pigra eats a leaf of Cirsium setosum. Asterisks mark the beetle location; (D)—Cleonis Cleonis pigra on the roadside; (E)—Cleonis pigra on the seashore. All photos: S. Volovnik. pigra on the roadside; (E)—Cleonis pigra on the seashore. All photos: S. Volovnik.

3.2.4. Life Cycle InIn Ukraine, adults were recorded from late Apri Aprill (Eupatoria, (Eupatoria, Yevpatoria, Yevpatoria, East Crimea) onwards. InIn the Czech Republic, thethe firstfirst adultsadults areare foundfound earlier,earlier, inin MarchMarch [[65].65]. In the northeastern USA, adults appear later, inin MayMay [[82].82]. ActiveActive imaginesimagines from thethe newnew generationgeneration werewere observedobserved fromfrom thethe secondsecond partpart ofof AugustAugust toto thethe endend ofof OctoberOctober (Podilsk,(Podilsk, OdessaOdessa Province).Province). Once, an active adult waswas foundfound asas latelate asas thethe beginningbeginning ofof DecemberDecember [[83].83]. InIn thethe spring,spring, thethe adultsadults feedfeed firstfirst onon thethe rosettesrosettes ofof thethe hosthost plantsplants andand thenthen gnawgnaw thethe youngyoung leavesleaves onon thethe shoots.shoots. Mating and oviposition occur from the end of AprilApril toto thethe endend ofof June.June. InIn thethe laboratory,laboratory, males males and and females females mated mated more more than than once once and and with with diff differenterent partners. partners. A mating A mating couple couple was observedwas observed by one by authorone author (S.V.) (S.V.) in copula in copula for one for hour. one hour. Females prefer to oviposit on plants with a larger root crown diameter and a higher number of shoots [84]. Cirsium arvense is dioecious, and according to Jung (cited by [79]), the roots of its female

Insects 2019, 10, 325 16 of 25

Females prefer to oviposit on plants with a larger root crown diameter and a higher number of shoots [84]. Cirsium arvense is dioecious, and according to Jung (cited by [79]), the roots of its female plants are attacked by Cleonis pigra three times more frequently than the roots of its male plants. A female of C. pigra searches for a suitable site for egg laying on the base of the host plant stem or root neck. Usually, this point is located partly under the soil surface. The female then makes a short tunnel through the soil with her rostrum, gnaws the pit into the cortex, and oviposits. Freshly laid eggs are matted, yellowish, light oval, 1.4–1.8 mm long, and 1.1–1.3 mm wide. The eggs are laid solitarily. The period of oviposition lasts up to the end of June [83]. Detailed descriptions of egg-laying behavior are provided in Cawthra [25]. In the laboratory, larvae hatched after 6–9 days (10–12 days according to [35]). In the field, young larvae were found from the beginning of April to mid-June. Larvae attacked the central part of the root where the vascular tissue is located. The mature larva coloration was milky white (Figure6A). Larvae gnawed tunnels downward, up to 4–5 mm in diameter. The tunnels may be straight or slightly curved (if this part of the root is curved as well) (Figure6B). All observed by one author (S.V.). In the process of larval development, a fusiform gall was formed on the root collar (Carduus hamulosus, Cirsium ukranicum) or on the taproot (C. hamulosus, C. ukranicum, Centaurea diffusa, and Centaurea odessana)[8] (Figure6C–E). The weevil also formed galls on Centaurea stoebe (syn. C. maculosa Lam.) [85]. The beginning of gall formation became visible at the end of the first instar of larval development. Root galls were typically located at a depth of 10–30 mm below ground level, although some were found up to 60 mm below ground level. As a rule, galls were located on the main root. Due to gall formation, growing larva stops moving and feeds by growing tissues around itself. This may result in the formation of a cavity with some dust (e.g., bits of vascular and other plant tissues). The larval stage lasts approximately 30 days [82]. Finishing its development, a larva gnaws a pupa chamber from 15–50 mm (in Centaurea odessana and Cirsium ukranicum, respectively) below the base of the root. The length of the chamber is 12–19 mm and the width is 6–7 mm (i.e., the chambers are slightly wider than the larval tunnel). This chamber may be situated below, above, or adjacent to the cavity of the tunnel. Before or after the construction of the chamber, larvae compacted bits of plant tissue and excrement as a type of cork above or below the chamber (Figure6F). This “cork” may be up to 10 mm in length. In another case, larvae compacted the dust on the walls of the chamber (Figure6G). Occasionally, several larvae were found mining in the same plant specimen. Therefore, their galls were formed one above another or/and side by side. A plant usually has one or two (and less frequently, up to six) galls. Typically, adjacent galls merge as they grow (but the larval tunnels and pupal chambers remain isolated). One such compound gall on C. ukranicum reached a size of 55 110 mm, whereas the × diameter of the base of the stem was 26 mm (Figure6H). In the spring, we found up to 10–15 larvae per plant specimen (Carduus hamulosus), but only 1–3 (maximum five) pupae and adults in the summer. There were dead larvae of 1st and 2nd instars found in one-third of dissected plants. The causes of their deaths are unknown (Figure6I). The first pupae were found at the beginning of July (5 July 1983). A young pupa is yellowish-orange (Figure6J–K). The last pupae were recorded up to the end of July. In their chambers, pupae and adults were typically situated head up and were rarely situated horizontally. Only once was a newly emerged adult in the pupal chamber located head down (Figure6L). A maximum of six and eight (Carduus hamulosus and Cirsium ukranicum, respectively) adults successfully finished their development in the same root. According to laboratory observations, the duration of the pupa stage is 14–21 days [83]. Insects 2019, 10, 325 17 of 25

Insects 2019, 10, x 17 of 25

Figure 6. Cleonis pigra life history. (A)—mature larva in the root crown of Cirsium ukranicum; (B)—the Figure 6. Cleonis pigra life history. (A)—mature larva in the root crown of Cirsium ukranicum;(B)—the curved tunnel, pupa chamber and adult; (C)—root gall of C. ukranicum; (D)—normal root of Centaurea curved tunnel, pupa chamber and adult; (C)—root gall of C. ukranicum;(D)—normal root of Centaurea didiffusaffusa (left)(left) andand gallgall on the same plant; ( E)—root gall on C. ukranicum ;(; (F)—adult in the larval chamber with corkcork above above the the chamber; chamber; (G)—larval (G)—larval tunnel tunnel and pupal and chamberpupal chamber on C. ukranicum on C. ;(ukranicumH)—a compound; (H)—a gallcompound on C. ukranicum gall on C.;( ukranicumI)—adult; and (I)—adult dead larva and indead a gall; larva (J)—young in a gall; ( pupaJ)—young of Cleonis pupa pigra of Cleonis;(K)—pupa pigra; begins(K)—pupa to sclerotize begins to from sclerotize its eyes fr andom its rostrum; eyes and (L)—occasionally, rostrum; (L)—occasionally, pupa and adult pupa may and be adult located may head be down.locatedAll head photos: down. S. All Volovnik. photos: S. Volovnik.

After emergence, adults remained in the gall untiluntil fully sclerotized, with an upperupper surface that was usuallyusually coveredcovered with with a a pollen-like pollen-like brownish brownish flush flush (Figure (Figure7A–C). 7A–C). The The adults adults chewed chewed their their way way out ofout the of pupalthe pupal chamber chamber within within the root the cortex. root First,cortex the. First, adult the gnawed adult outgnawed a small out roundish a small“window” roundish in“window” the wall. in Later, the wall. it gnawed Later, small it gnawed pieces small of root pieces cortex of near root the cortex apex near of its the rostrum. apex of The its rostrum. adult put The the tibiaadult and put tarsus the tibia of its and foreleg tarsus into of theits holeforeleg as itinto became the hole horizontally as it became elongated. horizontally This position elongated. provided This additionalposition provided support additional for gnawing. support The beetle for gnawing. clenched The the woodbeetle fibersclenched with the its mouthparts,wood fibers tiltedwith its headmouthparts, right and tilted left, its and head ripped right theand fiber. left, and Gnawing ripped went the fiber. on nonstop; Gnawing the went width on ofnonstop; the gnawed the width area of the gnawed area was increased, and the hole enlarged. After 30–40 min had elapsed, the weevils were leaving the gall (Figure 7D–E). Adults of the new generation emerged from the soil and fed on

Insects 2019, 10, 325 18 of 25 was increased, and the hole enlarged. After 30–40 min had elapsed, the weevils were leaving the gall Insects 2019, 10, x 18 of 25 (Figure7D–E). Adults of the new generation emerged from the soil and fed on the rosettes of young Cirsium. the rosettesAt thatof young stage, Cirsium. the adult At can that eat stage, up to 120the mgadult of can the leafeat up tissue to 120 at once mg (60%of the per leaf primary tissue at weight once (60%of the per body). primary weight of the body). Imagines hibernated outside outside of of host plants, most likely in the top layer of soil. Lindenberg [69] [69] wrote thatthat larvaelarvae sometimessometimes go go from from the the root root to to the the stem stem of theof the Scotch Scotch thistle thistle (Onopordum (Onopordum) where) where they theyfinish finish their their development. development. We neverWe never recorded recorded such such cases. cases. Hence, Hence,C. pigraC. pigrais ais univoltine a univoltine species species in inUkraine, Ukraine, and and across across Europe Europe and and North North America. America. There There is only is oneonly Communication one Communication on the on life the history life historyof C. pigra of C.near pigra the near eastern the eastern limits oflimits its area of its [86 area], where [86], wher the lifee the cycle life iscycle diff erent.is different. According According to these to theseauthors, authors, in Heilongjiang in Heilongjiang province province (northeastern (northea China),stern China), newly emergednewly emerged adults occuradults in occur late Augustin late Augustand lay and eggs lay in eggs early in September. early September. Larvae Larvae of the of second the second generation generation pupate, pupate, and and the pupaethe pupae pass pass the thewinter winter outside outside of the of hostthe host plants, plants, near near its roots. its roots. Adults Adults of the of nextthe next generation generation occur occur in mid-May. in mid-May.

Figure 7. 7. CleonisCleonis pigra pigra lifelife history history and and biotic biotic interactions. interactions. (A)—The (A)—The newly newly emerged emerged adult adulthas light- has colored,light-colored, soft integuments; soft integuments; (B)—later, (B)—later, integuments integuments become become more solid more and solid reddish-brown; and reddish-brown; (C)— finally,(C)—finally, the adult the adult takes takes on its on normal its normal grey grey color; color; (D ()—theD)—the emergence emergence of of the the adult adult from from the the stem stem of of Cirsium ukranicum. Time:Time: 12:19; 12:19; ( (EE)—the)—the emergence emergence of of the the adult adult from from the the stem stem of of CirsiumCirsium ukranicum. ukranicum. Time: 12:53; ( F)—ants)—ants ( (Lasius)) attack attack the the weevil weevil on on Cirsium ukranicum ;;( (G)—the body of a a dead dead beetle beetle infectedinfected by by fungi; fungi; ( (H)—the)—the larva larva of of C. pigra and the larva of a fly fly ( Cheilosya)) in in a root gall on Cirsium ukranicum.. All All photos: photos: S. S. Volovnik. Volovnik.

3.2.5. Biotic Biotic Interactions Interactions In the middle of May 1982, S. Volovnik found many dry fragments of C. pigra (mostly bodies without abdomens) on the coastal sands near Popi Popivkavka in western Crimea. Apparently, Apparently, these beetles were pecked by water birds. There There were were two two pellets pellets of of gulls gulls or or othe otherr Laridae Laridae (Aves, (Aves, Charadriiformes). Charadriiformes). The pellets consisted of remnants of 10 and 17 specimens of C. pigra. SometimesSometimes (usually (usually in in spring), spring), the adults of C. pigra are rather numerous. In open habitats (coasts, steppes, and semideserts), these relatively large beetles become the prey of carnivorous animals. In the stomach of steppe birds, namely, the great bustard (Otis tarda Linnaeus, 1758) and the little bustard (Tetrax tetrax (Linnaeus, 1758) (Aves, Otididae)), as many as nine and eight specimens of C. pigra, respectively, were recorded

Insects 2019, 10, 325 19 of 25 the adults of C. pigra are rather numerous. In open habitats (coasts, steppes, and semideserts), these relatively large beetles become the prey of carnivorous animals. In the stomach of steppe birds, namely, the great bustard (Otis tarda Linnaeus, 1758) and the little bustard (Tetrax tetrax (Linnaeus, 1758) (Aves, Otididae)), as many as nine and eight specimens of C. pigra, respectively, were recorded [87]. In other seasons and other habitats, beetles of C. pigra are only the accidental prey of many terrestrial vertebrates, especially birds, (e.g., [88,89]), and crabronid wasps from the genus Cerceris L. [90,91]. In cavities of the roots of Centaurea, the ant Lasius alienus (Förster, 1850) (Formicidae, Hymenoptera) and immature stages of C. pigra lived divided only by a thin wall, but the ants did not try to get their potential victims [92]. Occasionally, the roots of Cirsium ukranicum were surrounded by the nest of Lasius sp. This had no effect on the weevils, but ants attacked the weevils upon discovering them on the stems and foliage of the same plant specimen (Figure7F). Empty galls of Centaurea diffusa and Cirsium ukranicum became habitats for Lasius ants. Rarely, the pupae and adults of C. pigra were infected and destroyed by parasitic fungi [92] (Figure7G), but it is unknown how this infection occurred there—before or after the insect’s death. In laboratory experiments, a high air humidity results in the appearance of the fungus Beauveria bassiana (Bals.-Criv) Vull. on the body of the dead weevils C. pigra [93]. Sometimes, after dissecting the root galls of C. pigra, we found the cocoons of some parasitoid Hymenoptera (not identified). They passed the winter in the roots they parasitized. In northern Ukraine, up to 80% of C. pigra larva were eliminated by the parasitoid wasp Bracon discoideus Wesmael, 1838 (Hymenoptera: Braconidae) [69]. From larvae found in southeastern Scotland, seven specimens of the braconid wasp Bracon sphaerocephalus Szépligeti, 1901 [94] were reared. In the same region, 20%–60% of Cleonis galls were inhabited by the koinobiont endoparasitoid Acaenitus dubitator (Panzer, 1800) (Hymenoptera: Ichneumonidae) [95]. The parasitic wasps Vipio tentator Rossi, 1790 (Braconidae) and Aritranis fuscicornis Tschek, 1871 (Ichneumonidae) attacked late instar larvae in southern Europe [85]. Cawthra [25] also recorded indefinite Diptera and Nematodes as parasites on C. pigra larvae. One author (S.V.) found larval Cheilosia sp. (Diptera: Syrphidae) and larval C. pigra in the same root tunnel (Figure7H). Unfortunately, no progress was made to obtain an adult from the larval Diptera. Many of the Cheilosia spp. are known mainly as phytophagous, and its larvae often feed on plant tissues in the stems of Asteraceae [96–98]. Thus, Cheilosia can be a competitor for the larvae of C. pigra. Weevils are also infected by saprophagous gnats (Bradysia spp., Diptera, Sciaridae [84]). At times, the larvae of Cleonis pigra may coexist peacefully in a compound gall (see above) with the larvae of other gall-making insects that feed on anatomically different parts of the gall; namely, the root collar and root cortex [85]. In general, 14 species of phytophagous insects competed with C. pigra in the roots of Centaurea diffusa in southern and central Europe [84,99]. These species are separated in time and space. Müller [85] provided an example of such separation: he never found two niche competitors, C. pigra and Cyphocleonus achates at the same site. Our data differed greatly from that. Those two weevil species were in the same locality, which was the second plot where observations were performed (see Materials and Methods). Both species developed in the roots of C. diffusa. Sometimes, the plants infected by one of them were located within several meters of the plant infected with another. Thus, these species could coexist. Because C. achates is one of the main biological control agents in North America [100], it should be considered as a biological control agent elsewhere. Only one mature larva (C. pigra or C. achates, but not both) was recorded per spotted knapweed root. Rarely can two or three larvae be found in the same root [85]. Apparently, the space inside the roots of Centaurea diffusa is too limited to support the isolation of multiple larvae, and the larger larva can destroy the smaller larva. Perhaps our second plot (1) was abundant in food and/or (2) one or both weevil species newly settled there. Regardless, neither species was eliminated by direct competition or another ecological mechanism. In July, Cawthra [25] did not find C. pigra larvae in flowering host plants but did find them in non-flowering plants. This could mean that females prefer younger plants for egg laying. This is likely because surface layers of young plants are softer to gnaw for females or newly hatched larvae. Thus, Insects 2019, 10, 325 20 of 25 plants that began to grow earlier (before mating and egg laying) were more likely to avoid damage. As a result of larval development, the aboveground part of infested plants becomes significantly smaller and has fewer flowers, but the number of shoots increases [84,99]. The larval development, and probably, gall formation, both cause damage to the transport tissue (xylem). This results in the wilting of thistle (Cirsium arvense) and some reduction in the number of flowers. Nevertheless, xylem regenerates and attacked plants rarely die [77]. In spring, when host plants are still young, the damage may result in plant death.

3.2.6. What Are the Other Effects of the Weevil? Infested specimens of Silybum marianum tended to develop additional lateral roots in the soil surface to better absorb atmospheric water [32]. Because adventitious root buds formed on the taproot [101], damage caused by the larval activity of C. pigra and gall formation negatively affects the vegetative propagation of Cirsium. Along with the flower feeders (e.g., Larinus spp.), this can have the synergetic effect of biological control. In general, the activity of both C. pigra and Cyphocleonus achates has a minor impact on the number of Centaurea diffusa plants. There are plots where grass cover is practically absent, but annually, this plant is abundant. If the stem of Cirsium arvense breaks down (e.g., by the wind), the break is located above the root crown, and therefore, insects are safe there. The senesced adult plant C. diffusa can function as a tumbleweed. A damaged root crown breaks off by wind more easily than a healthy root crown. Therefore, beetle activity indirectly contributes to the dispersal of seeds of its host plant.

4. Conclusions The morphological features of immature Cleonis pigra (in both larval and pupal stages) are typical for the subfamily Lixinae. Some original larval morphological features (e.g., the presence of endocarina, the count and position of setae on the labrum, and the count of setae on mandibular) seem to be important features for generic study of the Cleonini. Keys for the determination of selected Cleonini species based on their larval and pupal characteristics are attached. Additionally, Dyar’s law was also used to estimate a number of larval instars of C. pigra. Moreover, the description of habitats, adult behavior, host plants, life cycle, and biotic interactions are discussed in detail here. Finally, it seems that such studies should improve the current knowledge of the weevil distributions and should provide practical guidance in the use of this group as a potential biological control agent.

Author Contributions: Conceptualization, J.S. and S.V.; data curation, J.S., S.V., R.G., R.S., and F.T.; funding acquisition, J.S. and R.G.; methodology, J.S., S.V., R.G., R.S., and F.T.; project administration, J.S. and S.V.; resources, J.S.; writing—original draft, J.S. and S.V.; writing—review and editing, R.G., R.S., and F.T. Funding: This research of JS was funded by the Czech Ministry of Agriculture (Mze CR)ˇ RO0418. Acknowledgments: We are sincerely grateful to V.P.Kolomiychuk (A.V.Fomin Botanical Garden, Taras Shevchenko National University of Kyiv) for identification of the plant species and to the entomologists of Schmalhausen Institute of Zoology, National Academy of Sciences of Ukraine, Kyiv: V.A. Korneyev and G. V. Popov—for identification of flies; A.G. Radchenko—for identification of ants. B. Korotyaev (Zoological Institute, Russian Academy of Sciences, St. Petersburg), A. V. Shavrin (Daugavpils University, Latvija), V. Yu. Nazarenko (Schmalhausen Institute of Zoology, Kyiv), Vasily Grebennikov (Canadian Food Inspection Agency, Ottawa, Canada), and Nidhi Sanwal (Indian Agricultural Research Institute, New Delhi) helped us with valuable information and literature. Conflicts of Interest: The authors declare no conflict of interest. Insects 2019, 10, 325 21 of 25

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